I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens
pET24a Vector. I inocculate my LB medium from an overnight culture, let the
culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one
night there is a band at 25 kDa in SDS-PAGE made with total cell protein.
When I try to separate cytosolic proteins from the rest and make another
SDS-PAGE with both fractions there is no band there.
I have two questions:
1. If the protein band is not the desired protein what can it be ?
2. If the band represents the protein, is it possible that it is proteolysed
within minutes ?